Exosomal MiR-21 Produced By Mechanical Ventilation Promotes Pulmonary Inflammatory Response Via Negative Regulation Of Notch2

Objective:Investigate the elevated miR-21 in lung tissue that induced by mechanical ventilation leads to pulmonary inflammation through exosomes,and determine whether the target of miR-21 is Notch2.Methods:The experiment was divided into three parts.Experiment 1: To investigate the increase of miR-21 expression in lung tissue which was induced by mechanical ventilation promotes pulmonary inflammation.Thirty-two c57 male mice were randomly divided into 4 groups:Ctrl;MV 2h;MV 6h;MV 12 h,eight mice per group.Intraperitoneal injection of 2% pentobarbital,and then mechanical ventilation through tracheotomy.The tidal volume was ventilated with 30 ml/Kg,and then they were humanely killed.The concentration of total protein?IL-6,TNF-? in BALF were measured by spectrophotometer and ELISA kits.Lung wet/dry ratio were measured;paraffin section and HE dyeing of the right middle lobe were for histopathology observation.The expression of Notch2 and Hes-1 was detected by Western blotting.The total RNA was extracted from the right lung wedge and detected miR-21 and Notch2 mRNA by RT-PCR.Experiment 2: The elevated miR-21 in lung tissue that induced by mechanical ventilation released through exosomes promoting inflammation.Accroding to experiment 1,MV 12 h was selected,and twenty-four c57 mice were randomly divided into 3 groups:Ctrl;MV 12h;GW4869+MV 12 h,eight mice per group.Mice in GW4869+MV 12 h were treated with GW4869 intravenously at a dose of 2.5?g/g,while Ctrl group and MV 12 h group were treated with an equal dose of 2.5%DMSO.One hour later they were ventilated 12 hours or not ventilated and sacrificed.The BALF was subjected to ultracentrifugation to obtain exosomes,which were identified by electron microscopy.Total RNA in exosomes was extracted and detected by RT-PCR.The concentration of total protein?IL-6 and TNF-? in BALF was detected.Lung wet/dry ratio were observed.Paraffin section and HE dyeing of the right middle lobe were for histopathology observation.The expression of Notch2 and Hes-1 was detected by Western blotting.The expression of Notch2 mRNA was detected by RT-PCR.Experiment 3:Study Notch2 mRNA is a downstream target gene of miR-21.The Raw264.7 cell line was randomly divided into the following groups:(1)miR-21 negative control group(NC);(2)miR-21 mimic group(miR-21 mimic);(3)miR-21 inhibitor group(miR-21 inhibitor).After 72 hours of transfection,the expression of Notch2 mRNA was detected by RT-PCR,and the expression of Notch2 protein was detected by Western blotting.Results:In the experiment 1,compared with the control group,the ventilation group showed alveolar dysfunction,inflammatory cell infiltration,alveolar wall thickening and hyperemia;and the alveolar wall thickening in MV 12 h group was more obvious.The alveolar morphology was normal and almost no inflammatory cell infiltration in control group.The wet/dry ratio of pulmonary,the total concentration and the cytokines IL-6 and TNF-? in BALF increased sharply in MV 12 h group.MV can increase the miR-21.MV can reduce the Notch2 mRNA and Notch2 and Hes-1 of lung tissue.In the MV 12 h group the changes was most significant.In the experiment 2,MV can lead to exosomes releasing into BALF,and the miR-21 in exosomes was significantly higher in MV 12 h compared with control group.The miR-21 in BALF was significantly decreased after being treated with GW4869.Compared with MV 12 h group,inflammatory cell infiltration,alveolar wall hyperemia and alveolar wall thickening in GW4869+MV 12 h group were significantly mitigated.The total protein content in BALF and the content of cytokines IL-6 and TNF-? in BALF decreased.The expression of Notch2 mRNA and protein in lung tissue increased,and the expression of Hes-1 protein increased.In the experiment 3,after transfection of miR-21 mimic,the expression of Notch2 mRNA and protein increased.And after transfection with miR-21 inhibitor,the expression of Notch2 mRNA and protein decreased.Conclusion:Mechanical ventilation induces the release of exosomes containing miR-21 and miR-21 promotes pulmonary inflammatory responses by negatively regulating Notch2.